SARS-CoV-2 infection and stem cells: Interaction and intervention.

The emergence of the novel severe acute respiratory coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread have created a global health emergency. The resemblance with SARS-CoV in spike protein suggests that SARS-CoV-2 employs spike-driven entry into angiotensin-converting enzyme 2 (ACE2)-expressing cells. From a stem cell perspective, this review focuses on the possible involvement of ACE2+ stem/progenitor cells from both the upper and lower respiratory tracts in coronavirus infection. Viral infection-associated acute respiratory distress syndrome and acute lung injury occur because of dysregulation of the immune response. Mesenchymal stem cells appear to be a promising cell therapy given that they favorably modulate the immune response to reduce lung injury. The use of exogenous stem cells may lead to lung repair. Therefore, intervention by transplantation of exogenous stem cells may be required to replace, repair, remodel, and regenerate lung tissue in survivors infected with coronavirus. Ultimately, vaccines, natural killer cells and induced-pluripotent stem cell-derived virus-specific cytotoxic T lymphocytes may offer off-the-shelf therapeutics for preventing coronavirus reemergence.

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat's weight (260-330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa's score, a larger infarct volume and a greater model success ratio were screened using the Longa's score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

Retrospective analysis on the efficacy of sunitinib/sorafenib in combination with dendritic cells-cytokine-induced killer in metastasis renal cell carcinoma after radical nephrectomy.

OBJECTIVE: Sunitinib/sorafenib (SU/SO), dendritic cells (DCs), or DC-cytokine-induced killer (CIK) could significantly prolong progression-free survival (PFS), 3-year overall survival (OS), or 5-year OS for patients with metastatic renal cell carcinoma (mRCC). We retrospectively analyzed the clinical efficacy between SU/SO combined with DC-CIK and SU/SO monotherapy in treating renal cell carcinoma (RCC) patients with metastasis after radical nephrectomy. MATERIALS AND METHODS: All patients (n = 34) with postoperative mRCC in our hospital from January 2009 to January 2014 were received either SU/SO monotherapy (Group 1, n = 15) or in combination with DC-CIK (Group 2, n = 19). A retrospective study was based on the primary endpoint (PFS) and secondary endpoint (OS). RESULTS: At a median follow-up of 19.5 months, in Group 2, as compared with in Group 1, the median PFS was significantly longer (28.0 vs. 11.0 months, P = 0.03). Moreover, the 3-year OS was higher (57.1% vs. 28.6%). The cases of progressive diseases (PDs) and deaths were less in Group 2 than that in Group 1 (PD: 8 vs. 9, deaths: 3 vs. 5); however, the cases of stable diseases were more (11 vs. 6). In addition, the 3-year OS was higher in SU + DC-CIK group than that in SO + DC-CIK group (63.36% vs. 50%). There was no significant difference for PFS between SO + DC-CIK group and SU single agent group. CONCLUSIONS: SU/SO with DC-CIK could significantly prolong the median PFS, improve the 3-year OS rate, prolong the 3-year OS. It is likely to be a new approach for mRCC after radical nephrectomy.

Validation of a preclinical animal model to assess brain recovery after acute stroke.

This study was to validate the animal model for the research in the stage of recovery and sequela of ischemic stroke. For its recognized many advantages and widespread applications, middle cerebral artery occlusion / reperfusion (MCAO/R) in Male Sprague-Dawley rats was chosen to be the foundation model. Then the weight of rats (260-330 g), the thread bolt type (2636/2838/3040/3043), the time of brain infarct (2/3 h) were tested to choose the larger infarct volume, higher Longa's score and model success rate through Longa's score and TTC staining. Finally, optimum condition of model was used in long period observing from 1 to 90 days after MCAO/R via five assessment of sensorimotor functions and TTC staining. The results showed that the optimal rat model of cerebral infarction in the stage of recovery and sequela of ischemic stroke maybe the model rats which were 300 g weight and MCAO with 3040 line-lock for 3 h before reperfusion. In these conditions, the Longa's score was 2.1 ±â€¯0.2, and infarct volume was 23.0 ±â€¯2.4%. The sensorimotor functional test of bilateral asymmetry had significant difference from 1 to 90 days, the test of grid-walking had significant difference from 1 to 60 days, while other tests had significant difference only at 1 day after MCAO/R. In conclusion, 3040-300 g-3 h was the most appropriate condition, and the appropriate index of sensorimotor functions were bilateral asymmetry and grid-walking test.

Effects of human umbilical cord-derived mesenchymal stem cells on hematologic malignancies.

Mesenchymal stem cells (MSCs) have been used in hematopoietic stem cell transplantation for years. However, the safety of MSCs applied in various types of hematologic malignancy has not been comprehensively explored. In the present study, the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on six representative hematologic malignancy cell lines were explored, including leukemia, multiple myeloma and lymphoma cells. Direct and indirect co-culture models were established, and cell proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester staining. A cytometric bead array cytokine kit was used to quantify cytokines. The expression of interleukin (IL)-6 receptor elements on tumor cells was detected by reverse transcription-polymerase chain reaction and flow cytometry, and the effects of exogenous IL-6 on cell proliferation were determined using a Cell Counting kit-8 assay. The results demonstrated that hUC-MSCs inhibited the proliferation of most of the cell lines examined (THP-1, HL-60, K562 and RPMI-8226), but promoted the proliferation of Raji cells. In addition, hUC-MSCs secreted abundant IL-6, promoted the secretion of IL-10 by RPMI-8226 and Raji cells, and inhibited the secretion of tumor necrosis factor-α by THP-1 cells. These data indicate a varied effect of hUC-MSCs on various types of hematologic malignancy, including distinct mechanisms of cell-to-cell contact and cytokines. Researchers applying hUC-MSCs in lymphoma should be aware of a potential tumor growth-promoting effect.

Simultaneous harvesting of endothelial progenitor cells and mesenchymal stem cells from the human umbilical cord.

The human umbilical cord (UC) is usually discarded as biological waste. However, it has attracted interest as a source of cells including endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs), which have demonstrated enormous potential in regenerative medicine. The present study describes a convenient protocol that has been developed to sequentially extract these two cell types from a single UC. EPCs which had properties of progenitor cells were successfully isolated from the UC vein. These cells had cobble-shaped morphology and expressed Flt-1, KDR, VE-cadherin, von Willebrand factor and CD31 mRNA, in addition to CD73, CD105 and vascular endothelial growth factor receptor-2. In addition to absorbing fluorescent-labeled acetylated low density protein and binding to fluorescein isothiocyanate-UEA-l, they were able to form vascular tube-like structures on Matrigel. Typical fibroblast-like cells, which were isolated from the Wharton's jelly, were confirmed to be MSCs by their expression of CD73, CD90 and CD105, and their ability to differentiate into adipocytes and osteoblasts. Thus, the human UC-derived cells may be suitable for use in tissue engineering and cell therapy.

Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells.

Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1'-dioctadecyl-3,3,3',3'-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration.

[Inhibition of sunitinib on the expressions of PD-L1 and PD-L2 of mouse bone marrow-derived dendritic cells].

OBJECTIVE: To observe the changes of programmed death-1 ligand 1 (PD-L1) and PD-L2 expressions on mouse bone marrow-derived dendritic cells (DCs) stimulated by sunitinib. METHODS: DCs were randomly divided into four groups which were treated with sunitinib (100, 200, 300 ng/mL) and dimethylsulfoxide (DMSO), respectively. After 48 hours, PD-L1 and PD-L2 expression levels were analyzed by flow cytometry. RESULTS: Compared with the control group, the expression of PD-L1 on mature DCs (mDCs) and all DCs [including mature DCs and immature DCs (imDCs)] was significantly down-regulated in sunitinib treatment groups. The PD-L1 expression percentages of imDCs, mDCs and DCs were significantly reduced in sunitinib treatment groups; the percentage of mDCs expressing PD-L2 also dropped in all treatment groups, and the percentage of DCs expressing PD-L2 decreased in 100 and 300 ng/mL sunitinib treatment groups. CONCLUSION: Sunitinib can significantly reduce the expressions of PD-L1 and PD-L2 on mouse DCs.

Proteomic analysis of bladder cancer indicates Prx-I as a key molecule in BI-TK/GCV treatment system.

In order to understand the molecular mechanisms of Bifidobacterium infantis thymidine kinase/nucleoside analogue ganciclovir (BI-TK/GCV) treatment system which was proven to exhibit sustainable anti-tumor growth activity and induce apoptosis in bladder cancer, a proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. 192 down-regulated and 210 up-regulated proteins were identified after treatment with BI-TK/GCV system in Sprague-Dawley (SD) rats. Western blot analysis and immunohistochemistry analysis confirmed that Peroxiredoxin-I (Prx-I) was significantly down-regulated in bladder cancer after treatment. Prx-I silencing by transfection of Prx-I shRNA significantly suppressed growth, promoted apoptosis and regulated the cell cycle in T24 cells and reduced the phospho-NF-κB p50 and p65 protein expression which revealed the links between Prx-I and NF-κB pathway implied by Ingenuity pathway analysis (IPA). These findings yield new insights into the therapy of bladder cancer, revealing Prx-I as a new therapeutic target and indicating BI-TK/GCV system as a prospective therapy by down-regulation of Prx-I through NF-κB signaling pathway.

Decreased expression of NPRL2 in renal cancer cells is associated with unfavourable pathological, proliferation and apoptotic features.

The tumor suppressor gene nitrogen permease regulator-like 2(NPRL2) NPRL2 expressed obviously in many normal human tissues, but reduced in expression in many human tumors significantly. In this study, we detected the expression of NPRL2 in 78 clear cell renal cell carcinoma (ccRCC) by immunohistochemistry and correlated it with clinicopathological parameters. Meanwhile, the function of NPRL2 in human ccRCC was further explored after transfected recombinant expressing plasmids pEGFP-N1-NPRL2 into human renal cancer 786-0 cells. NPRL2 protein showed high expression in 67 of 78 cases of adjacent normal tissues (85.9 %), which was significantly higher than that in ccRCC tissues (23/78, 29.5 %). Clinic pathological analysis showed that NPRL2 expression was significantly correlated with histological grade (P = 0.044), TNM stage (P = 0.025) and lymph node metastasis (P = 0.028). MTT assay demonstrated that NPRL2 could obviously inhibit renal cancer cell proliferation. Flow cytometric analysis revealed that NPRL2 could induce renal cancer cells apoptosis and arrest the cell cycle in G0/G1 phase. In conclusion, NPRL2 is closely correlated to unfavourable pathological, proliferation and apoptotic features in ccRCC.

Infusion of Trx-1-overexpressing hucMSC prolongs the survival of acutely irradiated NOD/SCID mice by decreasing excessive inflammatory injury.

A protective reagent for ARI should have the ability to repair injured tissue caused by radiation and prevent continuous damage from secondary risk factors. Trx-1 was explored as a candidate therapy for ARI, as it scavenges reactive oxygen species, regulates cell growth and differentiation, participates in immune reactions, and inhibits apoptosis by acting inside and/or outside cells. Trx-1 can also decrease excessive inflammation in ARI by regulating the creation of inflamed media, by inhibiting the activation of complement, and by reducing the chemotaxis, adhesion, and migration of inflammatory cells. As effectively and stably expressing exogenous genes in the long term and regulating immune inflammation and tissue repair, MSC are a good choice for Trx-1 gene therapy. In this study, Trx-1-overexpressing hucMSC-Trx-1 were obtained by adenoviral vector-mediated infection. We first measured the redox capacity of hucMSC-Trx-1 with an antioxidant capacity (T-AOC) assay, a hydrogen peroxide (H2O2) content determination assay in vivo, a H2O2-induced oxidation hemolysis assay, and a lipid peroxidation assay in vitro. Then, we measured survival time, the protection of the hematopoietic system, and the regulation of inflammation in important organs in three treatment groups of NOD/SCID mice (treated with hucMSC-Trx-1, with hucMSC, and with saline) that were exposed to 4.5 Gy (60)Co-γ-ray radiation. The hucMSC-Trx-1 group achieved superior antioxidation results, protecting bone marrow hematopoietic stem cells (Lin(-)CD117(+): hucMSC-Trx-1 vs. hucMSC, P<0.05; hucMSC-Trx-1 vs. NS, P<0.01), promoting the formation of red blood cells and hemoglobin (hucMSC-Trx-1 vs. hucMSC or NS, P<0.05), reducing inflammation and damage in important organs (Bone marrow and lung: hucMSC-Trx-1 vs. NS, P<0.01; hucMSC-Trx-1 vs. hucMSC, P<0.05. Liver and intestine: hucMSC-Trx-1 vs. NS, P<0.05; hucMSC-Trx-1 vs. hucMSC, P<0.05), and prolonging survival (hucMSC-Trx-1 vs. hucMSC or NS, P<0.01). Therefore, hucMSC-Trx-1 combines the merits of gene and cell therapy as a multifunctional radioprotector for ARI.

Proteomic analysis of bladder cancer by iTRAQ after Bifidobacterium infantis-mediated HSV-TK/GCV suicide gene treatment.

In our previous studies, we constructed the Bifidobacterium infantis thymidine kinase/nucleoside analogue ganciclovir (BI-TK/GCV) system, which was proven to have a sustainable antitumor activity in an in vivo bladder cancer rodent model. In this article, a proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ) and followed by liquid chromatography-tandem mass spectrometry was used to understand the molecular mechanisms of this system. iTRAQ identified 192 downregulated and 210 upregulated proteins after treatment with BI-TK/GCV in Sprague-Dawley rats. Downregulations of proliferating cell nuclear antigen (PCNA), pyruvate kinase isozymes M2 (PKM2), hexokinase 1 (HXK-1), 6-phosphofructokinase (PFK-B), and cell surface glycoprotein (CD146) in bladder cancer after treatment were confirmed by Western blot analysis and validated by immunohistochemistry. Furthermore, the networks of cancer proliferation associated with PCNA, glycolysis associated with PKM2, HXK-1, and PFK-B, and invasion associated with CD146 were illustrated using Ingenuity Pathway Analysis. This study represents the successful application of iTRAQ technology to reveal the molecular mechanisms of BI-TK/GCV treatment system and provides the theoretical support for the effectiveness of our successful treatment system.

[Effects of human umbilical cord mesenchymal stem cells on the proliferation of hematopoietic malignancies].

OBJECTIVE: To study the effects of human umbilical cord mesenchymal stem cells (UC-MSCs) on proliferation of eight tumor cell lines from leukemia, lymphoma and multiple myeloma in vitro. METHODS: Tumor cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). UC-MSCs were co-cultured with tumor cells at different ratios as experimental groups, meanwhile, tumor cells were cultured without UC-MSCs as control groups. After three days, mean fluorescence intensity and cell division generations of the tumor cells were measured by flow cytometry. RESULTS: UC-MSCs inhibited the proliferation of HL60, THP1, K562 and RPMI8226 cell lines, but promoted the proliferation of Raji and NCIH929 cell lines. UC-MSCs promoted the proliferation of Jurkat cells only at 1:1 ratio; as for U937 cells, UC-MSCs inhibited the proliferation at 2:1 ratio (UC-MSCs: U937), promoted proliferation at 1:4 and 1:16, whereas had no obvious effect at 1:1. CONCLUSION: UC-MSCs have different effects on the proliferation of different hematopoietic tumor cell lines. They have no promoting effects on four leukemic cell lines, but have bidirectional (promotion/inhibition) effects on lymphoma and multiple myeloma cell lines. The U937 cell line may serve as a good model for the mechanism study of this contradictory phenomenon.